Mass spectrometry has advanced over the last few decades to the point where it is one of the most broadly applicable analytical tools for detection and characterization of a wide class of molecules. Mass spectrometric analysis is applicable to almost any species capable of forming an ion in the gas phase, and, therefore, provides perhaps the most universally applicable method of quantitative analysis. In addition, mass spectrometry is a highly selective technique especially well suited for the analysis of complex mixtures of different compounds in varying concentrations. Further, mass spectrometric methods provide very high detection sensitivities, approaching tenths of parts per trillion for some species. As a result of these beneficial attributes, a great deal of attention has been directed over the last several decades at developing mass spectrometric methods for analyzing complex mixtures of biomolecules, such as peptides, proteins, carbohydrates and oligonucleotides and complexes of these molecules.
To be detectable via mass spectrometric methods, a compound of interest must first be converted into an ion in the gas phase. Accordingly, the ion formation process significantly impacts the scope, applicability, efficiency and limitations of mass spectrometry. Conventional ion preparation methods for mass spectrometric analysis are largely unsuitable for high molecular weight compounds, such as biomolecules. For example, vaporization by sublimation and/or thermal desorption is unfeasible for many high molecular weight biomolecules because these species tend to have negligibly low vapor pressures. Ionization methods based upon desorption processes, on the other hand, have proven more effective in generating ions from thermally labile, nonvolatile compounds. In these methods, a sample is subjected to conditions resulting in emission of ions from solid or liquid surfaces or generation of ions via complete evaporation of charged droplets.
Over the last few decades, two desorption based ion preparation techniques have been developed that are particularly well suited for the analysis of large molecular weight compounds: (1) matrix assisted laser desorption and ionization—mass spectrometry (MALDI-MS) and (2) electrospray ionization—mass spectrometry (ESI-MS). MALDI and ESI ion preparation methods have profoundly expanded the role of mass spectrometry for the analysis of nonvolatile high molecular weight compounds including many compounds of biological interest. These ionization techniques generally provide high ionization efficiencies (ionization efficiency=(ions formed)/(molecules consumed)) and have been demonstrated to be applicable to biomolecules with molecular weights exceeding 100,000 Daltons.
In MALDI, analyte is integrated into a crystalline organic matrix and irradiated by a short (≈10 ns) pulse of laser radiation at a wavelength resonant with the absorption band of the matrix molecules. This process results in rapid formation of a gas phase plume wherein analyte molecules are entrained and ionized via gas-phase proton transfer reactions. MALDI ion formation generally produces ions in singly and/or doubly charged states. Fragmentation of analyte molecules during vaporization and ionization, however, limits the applicability of MALDI for some samples, and the sensitivity of the technique is known to depend on sample preparation methodology and the surface and bulk characteristics of the site irradiated by the laser. As a result, MALDI-MS analysis is primarily used to identify the molecular masses of components of a sample and yields little information pertaining to the concentrations or molecular structures of materials analyzed. Further, MALDI ion sources are generally not directly compatible with systems useful for online sample purification prior to ion formation, such as capillary electrophoresis and high performance liquid chromatography systems.
ESI is a widely used field desorption ionization method that generally provides a means of generating gas phase ions with little analyte fragmentation [Fenn et al., Science, 246, 64-70 (1989)]. Furthermore, ESI is directly compatible with on-line liquid phase separation techniques, such as high performance liquid chromatography (HPLC) and capillary electrophoresis systems. In ESI, a solution containing a solvent and an analyte is pumped through a capillary orifice maintained at a high electrical potential and directed at an opposing plate provided near ground. The electric field at the capillary tip charges the surface of the emerging liquid and results in a continuous or pulsed stream of electrically charged droplets. Subsequent evaporation of the solvent from charged droplets promotes formation of analyte ions from species existing as ions in solution. Polar analyte species may also undergo desorption and/or ionization during the electrospray process by associating with cations and anions in solution. A number of other useful field desorption methods using electrically charged droplets have been developed in recent years that are also capable of preparing ions from non-volatile, thermally liable, high molecular weight compounds. These techniques differ primarily in the physical mechanism in which droplets are generated and electrically charged, and include aerospray ionization, thermospray ionization and the use of pneumatic nebulization.
In contrast to MALDI, ions produced by field desorption methods employing charged droplets typically generate analyte ions populating a number of different multiply charged states, including highly charged states. Mass spectra obtained using these techniques, therefore, may comprise a complex amalgamation of peaks corresponding to a distribution of multiply charged states for each analyte species in a sample. In some cases, mass spectra obtained using these techniques have too many overlapping peaks to allow effective discrimination and identification of the components of a sample comprising a complex mixture of analytes. Accordingly, the formation of analyte ions populating a relatively a large number of different multiply charged states limits the applicability of field desorption ionization methods employing electrically charged droplets for analysis of complex mixtures, such as samples obtained from cell lysates.
Over the last decade, various computational and experimental approaches for expanding the utility of ESI-MS techniques for the analysis of complex mixtures of biopolymers have been pursed. One approach uses computer algorithms that transform experimentally derived multiply charged ESI spectra to “zero charge” spectra [Mann et al., Anal. Chem., 62, 1702 (1989)]. While transformation algorithms take advantage of the precision improvement afforded by multiple peaks attributable to the same analyte species, spectral complexity, detector noise and chemical noise often result in missed analyte peaks and the appearance of false, artifactual peaks. The utility of transformation algorithms for interpreting ESI-MS spectra of mixtures of biopolymers may be substantially improved, however, by manipulating the charge-state distribution of analyte ions produced in ESI and/or by operating under experimental conditions providing high signal to noise ratios [Stephenson and McLucky, J. Mass Spectrom. 33, 664-672 (1998)]. Another approach to reducing the complexity of ESI-MS spectra of mixtures of biopolymers involves operating the electrospray ionization ion source in a manner that lowers and/or controls the net number of charge-states populated for a particular analyte compound. A variety of methods of charge reduction have been attempted with varying degrees of success.
Griffey et al. report that the charge-state distribution of analyte ions produced by ESI may be manipulated by adjusting the chemical composition of the solution discharged by the electrospray [Griffey et al., J. Am. Soc. Mass Spectrom., 8, 155-160 (1997)]. They demonstrate that modification of solution pH and/or the abundance of organic acids or bases in a solution may result in ESI-MS spectra for oligonucleotides primarily consisting of singly and doubly charged ions. In particular, Griffey et al. report a decrease in the average charge-state observed for the electrospray of solutions of a 14 mer DNA molecule from −7.2 to −3.8 upon addition of ammonium acetate to achieve a concentration of approximately 33 mM. Although in some cases altering solution conditions appears to improve the ease in which ESI spectra are interpreted, these techniques do not allow selective control over the distribution of charge states accessed for all species present in solution. In addition, manipulation of solution phase composition may also generate unwanted effects, such as compromising ionization and/or transmission efficiencies in the electrospray ionization process.
An alternative approach for controlling the charge-state distributions of analyte ions produced by ESI is involves the use of gas phase chemical reactions of reagent ions to reduce the ionic charges of droplets and/or analyte ions generated upon electrospray discharge. This approach has the advantage of at least partially decoupling ionization and charge reduction processes in a manner having the potential to provide substantially independent control of charge-state distribution. Independent control of charge reduction is beneficial as it provides flexibility in selecting the sample composition (e.g. pH, buffer concentration, ionic strength etc.) and the ESI operating conditions.
To achieve a reduction in the charge-state distribution generated in the electrospray discharge of a solution containing a mixture of proteins, Ogorzalek et al. merged the output of an electrospray discharge with a stream of reagent ions generated by an externally housed Corona discharge [Ogorzalek et al., J. Am. Soc. Mass Spectrom., 3, 695-705 (1992)]. Ogorzalek et al. observed a decrease in the most abundant cation observed in the electrospray discharge of solutions containing equine heart cytochrome c from a charge state of +15 to a charge state of +13 upon merging a stream of anions formed via corona discharge with the output of an ESI source operating in positive ion mode. While the authors report a measurable reduction in analyte ion charge state distribution, generation of a population consisting predominantly of singly and/or doubly charged ions was not achievable. Furthermore, the authors note that operation of the discharge at high discharge currents lead to a reduction in analyte ion signal equal to about two orders of magnitude. Regarding the potential application of their technique for “shifting charge state distributions,” the authors indicated “[o]ur experience suggests that the ion-ion reactions studied to date for this purpose are not as easy to control and appear to lead to greater signal losses than do ion-molecule reactions.”
U.S. Pat. No. 5,992,244 (Pui et al.) also report a method for neutralizing charged particles alleged to minimize particle losses to surfaces. In this method, charged droplets and/or particles are generated via electrospray and exposed to a stream of oppositely charged electrons and/or reagent ions flowing in a direction opposite to that of the electrospray discharge. The authors describe the use of a neutralization chamber with one or more corona discharges distributed along the housing for producing free electrons and/or ions for neutralizing the output of an electrospray discharge. Electrically biased, perforated metal screens or plates are positioned along the housing of the neutralization chamber between the corona discharges and a neutralization region to create a confined electric field to conduct reagent ions toward the electrospray discharge. In addition, Pui et al., describe a similar charged particle neutralization apparatus in which the corona discharge ion source is replaced with a radioactive source of ionizing radiation for generating reagent ions. In both methods, neutralization is reported to reduce wall losses and enhance neutral aerosol throughput to an optical detection region located downstream of the electrospray discharge.
U.S. Pat. Nos. 6,727,471 and 6,649,907 disclose methods, devices and device components providing charge reduction for field desorption ion sources using charged droplet, such as ESI and nebulization sources. In the patents, the output of a source of electrically charged droplets is directed through a field desorption-charge reduction chamber having a source of reagent ions. Reactions between charged droplets, analyte ions or both and oppositely charged reagent ions and/or electrons in the field desorption-charge reduction chamber reduces the charge state distribution of the analyte ions. The patents describe various means of improving high transmission efficiencies of analyte ions through the charge reduction chambers including use of a shield element surrounding the reagent ion source for substantially confining electric and/or magnetic fields generated by the reagent ion source, and use of a radioactive source of reagent ions. In addition, the patents provide various means for selectively adjusting the charge state distribution of reagent ions, including selective adjustment of the residence time of analyte ions and charged droplets in the field desorption-charge reduction chamber, selective adjustment of the voltage applied to a corona discharge reagent ion source, and selective adjustment of the flux of ionizing radiation generated by a radioactive ion source.
It will be appreciated from the foregoing that a need exists for devices and methods for regulating the charge-state distribution of ions generated by field desorption techniques to permit analysis of mixtures containing high molecular weight biopolymers via mass spectrometry. Particularly, methods, devices and device components are needed that provide selectively adjustable (i.e. tunable) charge reduction over a useful range of analyte ion charge states. Further, charge reduction methods and devices are needed that provide high analyte ion transmission and collection efficiencies required for sensitive mass spectrometric analysis.